Although a significant amount is known about bacterial infection, there is still much that remains to be elucidated about the proteins and other factors involved in this complex process. One protein complex that is important in bacterial infection is cytochrome bd oxidase. Cytochrome bd oxidases are membrane-bound protein complexes that function in cellular respiration and are found in eubacteria, eukaryotes and archaea. They are required for colonization of the gut by pathogenic Escherichia coli, survival of Brucella suis in macrophages, and protection of bacteria against nitric oxide, a component of the oxidative burst used by macrophages to kill engulfed pathogens. The cytochrome bd oxidase operon in E. coli and many other species of bacteria, including Salmonella enterica (salmonellosis), Vibrio cholerae (cholera), and Bordetella pertussis (whooping cough), encode an additional protein that has not been studied in cytochrome oxidase characterization to date. This short gene typically encodes a 35-50 amino acid protein predicted to contain a hydrophobic 1-helix and localize to the cell membrane. Based on its widespread conservation, its gene synteny with cytochrome bd oxidase genes, and its membrane localization, we hypothesize that this small protein is involved in cytochrome bd oxidase function in bacteria. To test our hypothesis, we plan to characterize two small proteins (YbgT and YccB) encoded within paralogous cytochrome bd oxidase operons in the bacterium E. coli and identify the role they play in cytochrome bd oxidase activity. To do this, the effects of altered levels of YbgT and/or YccB affect cytochrome bd oxidase activity will be determined in vivo (Aim 1) and in vitro (Aim 2). Numerous phenotypes have been identified for E. coli cells lacking wild-type levels of oxidase function, and bacteria with altered small proteins expression will be tested for similar phenotypes. In addition, cytochrome bd oxidase enzymatic activity and structure will be determined using membrane fractions collected from cells with altered small protein expression. We hypothesize that these small proteins likely act via protein- protein interactions with other membrane proteins, and these interactions will be investigated using biochemical purification and bacterial two-hybrid analysis (Aim 3). Finally, the amino acid sequences required for small protein function will be identified through mutagenesis followed by in vivo and in vitro protein characterization. This research will provide valuable new information about a well-studied protein complex, initiate a reevaluation of the role of small proteins in other protein complexes, and provide a new target molecule, the small transmembrane proteins, for the development of future antibiotics. PUBLIC HEALTH RELEVANCE: Cytochrome bd oxidase is an important component in bacterial survival in the host during pathogenesis. Although it is a well-studied protein complex, multiple lines of evidence suggest that small transmembrane proteins may be involved in cytochrome bd oxidase function. This research will identify the role of small proteins in the activity of this enzyme and provide a basis for developing new strategies for inhibiting cytochrome bd oxidase activity in pathogenic bacteria in the future. )